Synthesis of unsymmetrical dialkoxydiarylsilanes and diarylsilanediols from tetraalkoxysilane having a dioxasilepane unit.

The tetraalkoxysilane carrying a stable seven-membered dioxasilepane moiety and two trifluoroethoxy groups undergoes reliable iterative substitution of the two trifluoroethoxy groups by sequential treatment with different aryl Grignard reagents while keeping the seven-membered structure intact. The process results in the synthesis of unsymmetrical dialkoxydiarylsilanes and eventually diarylsilanediols after proper hydrolysis.

Post-embolization syndrome-like symptoms due to shedding of necrotic material of hepatocellular carcinoma into the bile duct following transcatheter arterial chemoembolization: an instructive case.

Fever, abdominal pain, and liver dysfunction are almost inevitable complications of transcatheter arterial chemo embolization (TACE) for hepatocellular carcinoma, but these symptoms may also be due to bile duct obstruction caused by shedding of necrotic tumor material into the bile duct. A 68-year-old man presented with persistent fever, liver dysfunction, and abdominal pain after TACE. Computed tomography revealed stone-like hyperdensities in the bile duct. Endoscopic retrograde cholangiopancreatography revealed these structures to be necrotic material from hepatocellular carcinoma. We believe this is an instructive case of an often overlooked situation.

Congenital anaemia associated with loss-of-function variants in DNA polymerase epsilon 1.

DNA polymerase epsilon (Pol ε), a component of the core replisome, is involved in DNA replication. Although genetic defects of Pol ε have been reported to cause immunodeficiency syndromes, its role in haematopoiesis remains unknown. Here, we identified compound heterozygous variants (p.[Asp1131fs];[Thr1891del]) in POLE, encoding Pol ε catalytic subunit A (POLE1), in siblings with a syndromic form of severe congenital transfusion-dependent anaemia. In contrast to Diamond-Blackfan anaemia, marked reticulocytopenia or marked erythroid hypoplasia was not found. Their bone marrow aspirates during infancy revealed erythroid dysplasia with strongly positive TP53 in immunostaining. Repetitive examinations demonstrated trilineage myelodysplasia within 2 years from birth. They had short stature and facial dysmorphism. HEK293 cell-based expression experiments and analyses of patient-derived induced pluripotent stem cells (iPSCs) disclosed a reduced mRNA level of Asp1131fs-POLE1 and defective nuclear translocation of Thr1891del-POLE1. Analysis of iPSCs showed compensatory mRNA upregulation of the other replisome components and increase of the TP53 protein, both suggesting dysfunction of the replisome. We created Pole-knockout medaka fish and found that heterozygous fishes were viable, but with decreased RBCs. Our observations expand the phenotypic spectrum of the Pol ε defect in humans, additionally providing unique evidence linking Pol ε to haematopoiesis.

LSD1 promotes the egress of hematopoietic stem and progenitor cells into the bloodstream during the endothelial-to-hematopoietic transition.

During embryonic development, primitive and definitive waves of hematopoiesis take place to provide proper blood cells for each developmental stage, with the possible involvement of epigenetic factors. We previously found that lysine-specific demethylase 1 (LSD1/KDM1A) promotes primitive hematopoietic differentiation by shutting down the gene expression program of hemangioblasts in an Etv2/Etsrp-dependent manner. In the present study, we demonstrated that zebrafish LSD1 also plays important roles in definitive hematopoiesis in the development of hematopoietic stem and progenitor cells. A combination of genetic approaches and imaging analyses allowed us to show that LSD1 promotes the egress of hematopoietic stem and progenitor cells into the bloodstream during the endothelial-to-hematopoietic transition. Analysis of compound mutant lines with Etv2/Etsrp mutant zebrafish revealed that, unlike in primitive hematopoiesis, this function of LSD1 was independent of Etv2/Etsrp. The phenotype of LSD1 mutant zebrafish during the endothelial-to-hematopoietic transition was similar to that of previously reported compound knockout mice of Gfi1/Gfi1b, which forms a complex with LSD1 and represses endothelial genes. Moreover, co-knockdown of zebrafish Gfi1/Gfi1b genes inhibited the development of hematopoietic stem and progenitor cells. We therefore hypothesize that the shutdown of the Gfi1/Gfi1b-target genes during the endothelial-to-hematopoietic transition is one of the key evolutionarily conserved functions of LSD1 in definitive hematopoiesis.

Immune response and protective efficacy of the SARS-CoV-2 recombinant spike protein vaccine S-268019-b in mice.

Vaccines that efficiently target severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent for coronavirus disease (COVID-19), are the best means for controlling viral spread. This study evaluated the efficacy of the COVID-19 vaccine S-268019-b, which comprises the recombinant full-length SARS-CoV-2 spike protein S-910823 (antigen) and A-910823 (adjuvant). In addition to eliciting both Th1-type and Th2-type cellular immune responses, two doses of S-910823 plus A-910823 induced anti-spike protein IgG antibodies and neutralizing antibodies against SARS-CoV-2. In a SARS-CoV-2 challenge test, S-910823 plus A-910823 mitigated SARS-CoV-2 infection-induced weight loss and death and inhibited viral replication in mouse lungs. S-910823 plus A-910823 promoted cytokine and chemokine at the injection site and immune cell accumulation in the draining lymph nodes. This led to the formation of germinal centers and the induction of memory B cells, antibody-secreting cells, and memory T cells. These findings provide fundamental property of S-268019-b, especially importance of A-910823 to elicit humoral and cellular immune responses.

Immunogenicity and protective efficacy of SARS-CoV-2 recombinant S-protein vaccine S-268019-b in cynomolgus monkeys.

The vaccine S-268019-b is a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S)-protein vaccine consisting of full-length recombinant SARS-CoV-2 S-protein (S-910823) as antigen, mixed with the squalene-based adjuvant A-910823. The current study evaluated the immunogenicity of S-268019-b using various doses of S-910823 and its vaccine efficacy against SARS-CoV-2 challenge in cynomolgus monkeys. The different doses of S-910823 combined with A-910823 were intramuscularly administered twice at a 3-week interval. Two weeks after the second dosing, dose-dependent humoral immune responses were observed with neutralizing antibody titers being comparable to that of human convalescent plasma. Pseudoviruses harboring S proteins from Beta and Gamma SARS-CoV-2 variants displayed approximately 3- to 4-fold reduced sensitivity to neutralizing antibodies induced after two vaccine doses compared with that against ancestral viruses, whereas neutralizing antibody titers were reduced >14-fold against the Omicron variant. Cellular immunity was also induced with a relative Th1 polarized response. No adverse clinical signs or weight loss associated with the vaccine were observed, suggesting safety of the vaccine in cynomolgus monkeys. Immunization with 10 µg of S-910823 with A-910823 demonstrated protective efficacy against SARS-CoV-2 challenge according to genomic and subgenomic viral RNA transcript levels in nasopharyngeal, throat, and rectal swab specimens. Pathological analysis revealed no detectable vaccine-dependent enhancement of disease in the lungs of challenged vaccinated monkeys. The current findings provide fundamental information regarding vaccine doses for human trials and support the development of S-268019-b as a safe and effective vaccine for controlling the current pandemic, as well as general protection against SARS-CoV-2 moving forward.

Pathogenic variants in the survival of motor neurons complex gene GEMIN5 cause cerebellar atrophy.

Cerebellar ataxia is a genetically heterogeneous disorder. GEMIN5 encoding an RNA-binding protein of the survival of motor neuron complex, is essential for small nuclear ribonucleoprotein biogenesis, and it was recently reported that biallelic loss-of-function variants cause neurodevelopmental delay, hypotonia, and cerebellar ataxia. Here, whole-exome analysis revealed compound heterozygous GEMIN5 variants in two individuals from our cohort of 162 patients with cerebellar atrophy/hypoplasia. Three novel truncating variants and one previously reported missense variant were identified: c.2196dupA, p.(Arg733Thrfs*6) and c.1831G > A, p.(Val611Met) in individual 1, and c.3913delG, p.(Ala1305Leufs*14) and c.4496dupA, p.(Tyr1499*) in individual 2. Western blotting analysis using lymphoblastoid cell lines derived from both affected individuals showed significantly reduced levels of GEMIN5 protein. Zebrafish model for null variants p.(Arg733Thrfs*6) and p.(Ala1305Leufs*14) exhibited complete lethality at 2 weeks and recapitulated a distinct dysplastic phenotype. The phenotypes of affected individuals and the zebrafish mutant models strongly suggest that biallelic loss-of-function variants in GEMIN5 cause cerebellar atrophy/hypoplasia.

Quantitative evaluation of hepatic integrin αvß3 expression by positron emission tomography imaging using 18F-FPP-RGD2 in rats with non-alcoholic steatohepatitis.

BACKGROUND: Integrin αvß3, which are expressed by activated hepatic stellate cells in non-alcoholic steatohepatitis (NASH), play an important role in the fibrosis. Recently, we reported that an RGD peptide positron emission tomography (PET) probe is useful as a predictor of hepatic fibrosis. Kinetic analysis of the RGD PET probe has been performed in tumours, but not in hepatic fibrosis. Therefore, we aimed to quantify hepatic integrin αvß3 in a model of NASH by kinetic analysis using 18F-FPP-RGD2, an integrin αvß3 PET probe. METHODS: 18F-FPP-RGD2 PET/CT scans were performed in control and NASH rats. Tissue kinetic analyses were performed using a one-tissue, two-compartment (1T2C) and a two-tissue, three-compartment (2T3C) model using an image-derived input function (IDIF) for the left ventricle. We then conducted correlation analysis between standard uptake values (SUVs) or volume of distribution (VT), evaluated using compartment kinetic analysis and integrin αv or ß3 protein expression. RESULTS: Biochemical and histological evaluation confirmed the development of NASH rats. Integrin αvß3 protein expression and hepatic SUV were higher in NASH- than normal rats. The hepatic activity of 18F-FPP-RGD2 peaked rapidly after administration and then gradually decreased, whereas left ventricular activity rapidly disappeared. The 2T3C model was found to be preferable for 18F-FPP-RGD2 kinetic analysis in the liver. The VT (IDIF) for 18F-FPP-RGD2, calculated using the 2T3C model, was significantly higher in NASH- than normal rats and correlated strongly with hepatic integrin αv and ß3 protein expression. The strengths of these correlations were similar to those between SUV60-90 min and hepatic integrin αv or ß3 protein expression. CONCLUSIONS: We have demonstrated that the VT (IDIF) of 18F-FPP-RGD2, calculated using kinetic modelling, positively correlates with integrin αv and ß3 protein in the liver of NASH rats. These findings suggest that hepatic VT (IDIF) provides a quantitative assessment of integrin αvß3 protein in liver.

In Vivo Multicolor Imaging with Fluorescent Probes Revealed the Dynamics and Function of Osteoclast Proton Pumps.

In vivo two-photon fluorescence imaging is a powerful modality to monitor cell dynamics in biomedical studies. To detect protein functions in living animals in real-time, fluorescent probes must show a quick response to the target function in specific tissues. Here, we developed a rhodamine-based small-molecule fluorescent probe called Red-pHocas (red pH-activatable fluorescent probe for osteoclast activity sensing) to reversibly detect the acidic environments for the spatiotemporal analysis of the function of osteoclast proton pumps. The introduction of electron-withdrawing N-alkyl substituents in the rhodamine spirolactam fluorophore remarkably increased the kinetics of the fluorescence response to acidic pHs, which allowed the rapid and reversible monitoring of acidic compartments and the analysis of the dynamics of osteoclast proton pumps during osteoclastic bone resorption. In vivo multicolor two-photon imaging using Red-pHocas in fluorescent reporter mice revealed that bone acidification occurred synchronously with the accumulation of proton pumps onto the bone surfaces. To our knowledge, this is the first study to demonstrate the direct involvement of osteoclast proton pumps in bone acidification under intravital conditions by means of an imaging probe.

Positive phototropism is accelerated in Biomphalaria glabrata snails by infection with Schistosoma mansoni.

Parasite-induced behavioral changes in their hosts favor to complete the lifecycle of parasites. Schistosome infection is also known to cause physiological changes in infected freshwater snail intermediate hosts. Here, we report, a novel phenomenon in which Schistosoma mansoni, a highly debilitating worm affecting millions of people worldwide, alters the phototropic behavior of Biomphalaria glabrata, the vector snail. S. mansoni-infection enhanced positive phototropism of vector snails and infected snails spent significantly more time in light. Possibly, these behavioral changes help the parasite to be released efficiently from the infected intermediate hosts, and to infect mammalian hosts.

Direct cell-cell contact between mature osteoblasts and osteoclasts dynamically controls their functions in vivo.

Bone homeostasis is regulated by communication between bone-forming mature osteoblasts (mOBs) and bone-resorptive mature osteoclasts (mOCs). However, the spatial-temporal relationship and mode of interaction in vivo remain elusive. Here we show, by using an intravital imaging technique, that mOB and mOC functions are regulated via direct cell-cell contact between these cell types. The mOBs and mOCs mainly occupy discrete territories in the steady state, although direct cell-cell contact is detected in spatiotemporally limited areas. In addition, a pH-sensing fluorescence probe reveals that mOCs secrete protons for bone resorption when they are not in contact with mOBs, whereas mOCs contacting mOBs are non-resorptive, suggesting that mOBs can inhibit bone resorption by direct contact. Intermittent administration of parathyroid hormone causes bone anabolic effects, which lead to a mixed distribution of mOBs and mOCs, and increase cell-cell contact. This study reveals spatiotemporal intercellular interactions between mOBs and mOCs affecting bone homeostasis in vivo.

Transovarial persistence of Babesia ovata DNA in a hard tick, Haemaphysalis longicornis, in a semi-artificial mouse skin membrane feeding system.

Bovine piroplasmosis, a tick-borne protozoan disease, is a major concern for the cattle industry worldwide due to its negative effects on livestock productivity. Toward the development of novel therapeutic and vaccine approaches, tick-parasite experimental models have been established to clarify the development of parasites in the ticks and the transmission of the parasites by ticks. A novel tick-Babesia experimental infection model recently revealed the time course of Babesia ovata migration in its vector Haemaphysalis longicornis, which is a dominant tick species in Japan. However, there has been no research on the transovarial persistence of B. ovata DNA using this experimental infection model. Here we assessed the presence of B. ovata DNA in eggs derived from parthenogenetic H. longicornis female ticks that had engorged after semi-artificial mouse skin membrane feeding of B. ovata-infected bovine red blood cells. The oviposition period of the engorged female ticks was 21-24 days in the semi-artificial feeding. Total egg weight measured daily reached a peak by day 3 in all female ticks. Nested PCR revealed that 3 of 10 female ticks laid B. ovata DNA-positive eggs after the semi-artificial feeding. In addition, B. ovata DNA was detected at the peak of egg weight during oviposition, indicating that B. ovata exist in the eggs laid a few days after the onset of oviposition in the tick. These findings will contribute to the establishment of B. ovata-infected H. longicornis colonies under laboratory conditions.

Venestatin, a Ca++-binding protein from the parasitic nematode Strongyloides venezuelensis, is involved in the larval migration process.

The secretory EF-hand Ca++-binding proteins act as calcium signaling molecules for control of cell functions, but those proteins from parasitic helminths are poorly understood. Here, we have identified and characterized an EF-hand Ca++-binding protein from the rodent nematode, Strongyloides venezuelensis, termed 'venestatin', which is highly conserved in Strongyloides spp. Canonical two EF-hand domains and a signal peptide are present in venestatin. A gel mobility shift assay and Ruthenium red staining indicated that the recombinant venestatin possesses binding ability with Ca++ ions. Endogenous venestatin was seemingly localized in the hypodermis and gut of the worms and was found in the excretory-secretory products. Quantitative reverse transcription-PCR data showed that venestatin-specific transcript was upregulated in the parasitic stages of S. venezuelensis, and the upregulation occurred promptly after larval invasion through the host's skin, but not in the case of in vitro incubation. Immunization of mice with recombinant venestatin caused a 55% reduction in larval migration to the lungs, and lung hemorrhaging was mild compared with non-immunized groups, suggesting that anti-venestatin sera may interfere with larval migration from skin to lung. Our results suggest that venestatin is secreted from the hypodermis and gut of S. venezuelensis, and has pivotal roles in larval migration.

Rapid On-Site Evaluation by Endosonographers during Endoscopic Ultrasonography-Guided Fine-Needle Aspiration for Diagnosis of Gastrointestinal Stromal Tumors.

BACKGROUND/AIMS: Endoscopic ultrasonography-guided fine-needle aspiration (EUS-FNA) has been used to diagnose gastrointestinal submucosal tumors (SMTs). Although rapid on-site evaluation (ROSE) has been reported to improve the diagnostic accuracy of EUS-FNA for pancreatic lesions, on-site cytopathologists are not routinely available. Given this background, the usefulness of ROSE by endosonographers themselves for pancreatic tumors has also been reported. However, ROSE by endosonographers for diagnosis of SMT has not been reported. The aim of this study was to evaluate the diagnostic accuracy of EUS-FNA with ROSE by endosonographers for SMT, focusing on diagnosis of gastrointestinal stromal tumor (GIST), compared with that of EUS-FNA alone. METHODS: Twenty-two consecutive patients who underwent EUS-FNA with ROSE by endosonographers for SMT followed by surgical resection were identified. Ten historical control subjects who underwent EUS-FNA without ROSE were used for comparison. RESULTS: The overall diagnostic accuracy for SMT was significantly higher in cases with than without ROSE (100% vs. 80%, p=0.03). The number of needle passes by FNA with ROSE by endosonographers tended to be fewer, although accuracy was increased (3.3±1.3 vs. 5.9±3.8, p=0.06). CONCLUSIONS: ROSE by endosonographers during EUS-FNA for SMT is useful for definitive diagnosis, particularly for GIST.

Initial development of Babesia ovata in the tick midgut.

The initial development of Babesia ovata in the midgut of the vector tick Haemaphysalis longicornis has been demonstrated through in vitro and in vivo studies. Although the research on the partial developmental cycles of B. ovata in the tick midgut was performed in our previous study by using ticks fed on experimentally B. ovata-infected cattle, detailed information on the developmental stages of B. ovata in H. longicornis was limited. This report describes the sequential development of stages of B. ovata in an in vitro study using B. ovata-infected erythrocytes and tick midgut contents. The in vivo study also confirmed the developmental stages in the midgut contents of artificially B. ovata-infected ticks. In this observation, we have recognized the distinct forms of B. ovata developmental stages in the tick midgut; the aggregation forms and ray bodies with shorter spikes and light-stained cytoplasm were shown by Giemsa staining. The similarities and differences of the stages as compared to previous reports have been discussed.

A cysteine protease from Spirometra erinaceieuropaei plerocercoid is a critical factor for host tissue invasion and migration.

Sparganosis in humans caused by the plerocercoid larvae of Spirometra erinaceieuropaei is found worldwide, especially in Eastern Asia and the Far East. Previous studies have suggested that dissolution of plerocercoid body, plerocercoid invasion of host tissue, and migration are important processes for sparganosis progression. However, the mechanisms underlying these processes have yet to be determined. Here, we demonstrated the enzymatic property and involvement of a native 23kDa cysteine protease (Se23kCP), purified from plerocercoids, in sparganosis pathogenesis. Se23kCP is mature protease consisting of 216 amino acids and has a high sequence similarity with cathepsin L in various organisms. Se23kCP conjugated with N-glycans, which have a core fucose residue. Both cysteine and serine protease-specific activities were determined in Se23kCP and their optimal pHs were found to be different, indicating that Se23kCP has a wide range of substrate specificity. Se23kCP was secreted from tegumental vacuoles of the plerocercoid to host subcutaneous tissues and degraded human structural proteins, such as collagen and fibronectin. In addition, the plerocercoid body was lysed by Se23kCP, which facilitated larval invasion of host tissue. Our findings suggest that Se23kCP induces host tissue invasion and migration, and might be an essential molecule for sparganosis onset and progression.

Characterization and antiviral activity of a newly identified defensin-like peptide, HEdefensin, in the hard tick Haemaphysalis longicornis.

Tick defensins are antimicrobial peptides that play a major role in the innate immunity of ticks by providing a direct antimicrobial defense. In this study, we identified and characterized a defensin-like encoding gene, HEdefensin, from the expressed sequence tags (EST) database of hemolymph from the hard tick Haemaphysalis longicornis. Expression of the gene in whole adult ticks and in different organs was upregulated during blood feeding, though not after Langat virus (LGTV) challenge. A synthetic HEdefensin peptide demonstrated significant virucidal activity against LGTV but not against an adenovirus in co-incubation virucidal assays. Moreover, the RNAi-mediated gene silencing of HEdefensin did not significantly affect the virus titer as compared to the control group. The data reported here have established the in vitro virucidal activity of the peptide against LGTV. However, its role in the innate antiviral immunity of H. longicornis remains to be explored, and further studies are needed to fully evaluate the potential biological activities of the peptide against bacteria, fungi or parasites.

Effects of a polysaccharide nanogel-crosslinked membrane on wound healing.

INTRODUCTION: Wound-dressing materials that promote wound healing while protecting wounds from infections are advantageous for clinical applications. Hence, we developed a cholesterol-bearing pullulan (CHP) nanogel that stimulated wound healing; however, it was mechanically weak and difficult to handle. Thus, the purpose of this study was to examine precisely the effects of a mechanically reinforced nanogel-crosslinked (NanoClik) membrane on wound healing. MATERIALS AND METHODS: NanoClik was prepared by mixing a thiol-terminated polyethylene glycol solution and an acryloyl group-modified CHP nanogel solution. A thin silicone sheet membrane, which was combined with NanoClik, was prepared. The NanoClick membranes and both positive and negative control membranes (collagen combined with silicone membrane and silicone membrane alone, respectively) were tested in vivo using a dorsal skin defect rat model. The rate and extent of wound healing was compared between groups after 7 and 14 days of implantation. RESULTS: In the NanoClik membrane group, the wound area was significantly reduced and neoepithelialization was promoted, compared with that observed in the other groups. In addition, extension and accumulation of collagen fibers were evident in the NanoClik membrane group. CONCLUSION: The NanoClik membrane is a strong candidate for use as an effective and safe wound-dressing material. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 544-550, 2017.